of a sedentary nematode, Heterodera schach- tii Schmidt, was achieved many years ago. (10). More recently, Murashige and Skoog. (MS) medium was used to ...
Journal of Nematology 22(3):395-398. 1990. © T h e Society of Nematologists 1990.
Reproduction of Globodera rostochiensis on Transformed Roots of Solanum tuberosum cv. Desiree A . KUMAR 1 AND J. M. S. FORREST 2 Abstract: T r a n s f o r m e d roots of the susceptible potato Solarium tuberosum L. cv. Desiree were inoculated with second-stage juveniles (]2) of Globodera rostochiensis pathotype R o l . Adult males emerged after 3 - 4 weeks and matings with females occurred. After 8 weeks gentle pressure on the eggs of maturing females released the J2 which were viable. Because this technique enables the production of vigorously growing roots with numerous laterals, it may be suitable for obtaining a high yield of sterile potato cyst nematodes.
Success with the sterile in vitro culture of a sedentary nematode, Heterodera schachtii Schmidt, was achieved many years ago (10). More recently, Murashige and Skoog (MS) medium was used to culture susceptible and resistant potato roots on which Globodera rostochiensis (Wollenweber) Behrens was reared (2). T h e use of transformed roots to rear H. schachtii and Meloidogyne javanica in vitro has opened up new possibilities for studying resistance genes against s e d e n t a r y n e m a t o d e s (8,13,14). T h e objective of this research was to determine whether G. rostochiensis could be cultured in vitro on transformed potato roots. MATERIALS AND METHODS
Transformed root cultures: Susceptible Solanum tuberosum L. cv. Desiree tubers were soaked in absolute ethanol for 5 minutes, peeled, and surface sterilized for 15 minutes in a 10% solution of Domestos, a commercial bleach (Unilever plc, London, UK) containing 7% sodium hypochlorite. They were then washed six times in sterile distilled water and finally immersed in liquid Murashige and Skoog medium (MS powder, Flow Laboratories, Irvine, UK) supplemented with 20 g sucrose per liter, adjusted to p H 5.8 (MS20) but without hormones (12). Several columnar sections were removed from the tubers using a sterile Received for publication 30 June 1989. 1 Cell Biologist, Department of Cellular and Molecular Genetics, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, Scotland. 2 Research Nematologist, Zoology Department, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, Scotland.
1-cm-d cork borer and sliced into disks 12 mm thick with a scalpel. T h e tuber disks were floated on 25 ml of MS medium containing Agrobacterium rhizogenes LBA 9402 carrying the Ri plasmid and binary vector pBin 19, obtained from Dr. J. Hamill (5). T h e bacterium had been grown overnight in YMB medium (6) containing 50 ~tg kanamycin/ml. After 30 minutes incubation with Agrobacterium, the tuber disks were transferred to the selection medium, MS20 medium solidified with 1% agar (Difco agar, Difco Laboratories, East Molesey, UK) containing 250 #g cefotaxime/ml (Roussel Laboratories, Uxbridge, UK) and 100 ~g kanamycin/ml (Sigma, Poole, UK). Cefotaxime was used to kill A. rhizogenes and kanamycin to select transformed roots. T h e plates were sealed with Nesco film (Nippon Shoji-Kaisha, Osaka, Japan) and incubated at 25 C in a 16-hour photoperiod. T u b e r disks were subcultured every 10 days onto the fresh selection medium. Developing roots that appeared after 4 weeks of culture were removed from the tuber disks and subcuhured on fresh selection medium at least five times at 10-day intervals. T h e vigorously growing hairy roots free from Agrobacterium were then selected from the young root tips, 1 cm long, and maintained on MS20 medium with 100/,g kanamycin/ ml.
Detection of neomycin phosphotransferase H (NPTII) activity: N P T II dot-blot assay was performed as previously described (11). T h e blot was exposed to X-ray film for 20 hours and the positive was developed and photographed.
Nematode sterilization and inoculation: 395
396 Journal of Nematology, Volume 22, No. 3, July 1990
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FIG. 1. Transformed roots growing from tuber disks ofSolanum tuberosum cv. Desiree. A) Four weeks after inoculation with A. rhizogenes on the selection medium. B) With many vigorously growing lateral hairy roots after subculture on MS20 medium.
Hatched second-stage juveniles (J2) of G. rostochiensis pathotype Rol were obtained as previously described (1). Cysts were presoaked for 7 days in tap water and then in potato root diffusate. T h e J2 that hatched within 24 hours were placed in a polystyrene syringe fitted with polyester mesh (3) to facilitate handling and stored for 1 week at 4 C in 50 ug streptomycin/ml water. Pieces of transformed potato root, ca. 1 cm long, were cut and transferred to nutrient agar (1.25% Davies agar with half strength MS20 medium) in petri dishes. After 3 days in antibiotic, the J2 were washed repeatedly in sterile distilled water, treated for 2 seconds with 0.01% (w/v) m e r c u r i c c h l o r i d e solution, and again washed thoroughly by flushing sterile distilled water through the syringe. T h e volume was reduced until the nematodes formed a dense suspension. Aliquots of 300-400J2 were drawn offand placed close to the roots with a sterile micropipet. Excess water was removed by blotting with sterile filter paper. T h e petri dishes were then sealed with Nesco film and placed in a dark incubator at 20 C. Root cultures were examined with a Kyowa stereomicroscope (40 x). After 7-8 weeks golden females were removed and cut open and J2
were released from the eggs by gentle pressure. Viable juveniles were motile soon after release or on examination 3 days later. RESULTS AND DISCUSSION
Inoculation of tuber disks of S. tuberosum cv. Desiree with A. rhizogenes LBA 9402 carrying both the plasmids Ri and Bin 19 produced vigorous root growth on the selection medium (Fig. 1A). These roots continued to grow rapidly when transferred to fresh MS20 m e d i u m lacking plant growth hormones but containing 100 ~g kanamycine/ml (Fig. 1B), demonstrating that they had been transformed by the cotransfer of the T-DNA from A. rhizogenes and the N P T II gene of the disarmed binary vector pBin 19 from A. tumefaciens, as previously reported (5). The kanamycinresistant phenotype of the hairy roots resulting from the expression of the N P T II gene was demonstrated by the N P T II dotblot assay. N P T II enzymatic activity was present in the transformed hairy roots and absent from the untransformed roots (not shown). Untransformed roots, which lack the gene N P T II, therefore were unable to grow on the medium containing kanamycin. Excised root pieces of transformed Desiree also grew well on MS20 medium
Reproduction of G. rostochiensis: Kumar, Forrest 397
FIc. 2. Globodera rostochiensis Rol on transformed roots. A) Golden females 7-8 weeks after inoculation. B) Brown females 9-10 weeks after inoculation.
with or without kanamycin, producing many side roots which were densely covered with root hairs (Fig. 1B). T h e J2 were able to penetrate the transformed roots and developed within them, as was reported by Mugnier (9). Males emerged after 3 - 4 weeks and moved across the agar to mate with expanding females which ruptured the root cortex and epidermis. After 7-8 weeks the females became golden in color (Fig. 2A). Areas of undifferentiated root growth often were found close to where the females fed (Fig. 2B). Browning of the roots also occurred. Most females contained large numbers of eggs. T h e nematode J2 released mechanically from the eggs were viable, as j u d g e d by their motility. As with H. schachtii on beet (10), the development of G. rostochiensis on Desiree was not adversely affected by transformation with A. rhizogenes. After 7 - 8 weeks golden females containing many viable eggs were p r e s e n t in a b u n d a n c e , as on untransformed roots (Forrest, unpubh ). Pathogenicity of these juveniles, however, was not tested, although results with H. schachtii suggest changes are unlikely (12). Because of the additional advantage of many vigorously growing root tips, this system should be suitable for the large-scale culture and storage of potato cyst nematode pathotypes under sterile conditions and for in vitro screening of large numbers of susceptible and resistant mutant lines in a pro-
gram of transposon mutagenesis designed to locate resistance genes in potato. LITERATURE CITED 1. Forrest, J. M. S., and L. A. Farrer. 1983. The response of the white potato cyst nematode Globodera pallida to diffusate from potato and mustard roots. Annals of Applied Biology 103:283-289. 2. Forrest, J. M. S. 1985. Changes in potato roots induced by potato cyst nematode (PCN). Annual Report of the Scottish Crop Research Institute, Dundee. P. 109. 3. Forrest, J. M. S. 1986. A modified syringe for processing second-stage juveniles of Globodera paUida for scanning electron microscopy. Nematologica 32: 122-123. 4. Forrest, J. M. S., W. M. Robertson, and E. W. Milne. 1989. Changes in the structure of amphidial exudate and the nature of lectin-labelling on the freshly hatched, invaded and emigrant second-stage juveniles of Globodera rostochiensis. Nematologica 12:337-341. 5. Hamill,J. D., A. Prescott, and C. Martin. 1987. Assessment of the efficiency of co-transformation of the T-DNA of disarmed binary vector derived from Agrobacterium tumefaciens and the T-DNA of A. rhizogenes. Plant Molecular Biology 9:573-584. 6. Hooykaas, P. J. J., P. M. Klapwijk, M. P. Nuti, R. A. Schilperoort, and A. R6rsch. 1977. Transfer of the Agrobacterium tumefaciens T I plasmid to virulent Agrobacteria and to Rhizobium ex planta. Journal of General Microbiology 98:477-484. 7. Johnson, R. N., and D. R. Viglierchio. 1969. Sugar beet nematode (Heterodera schachtii) reared on axenic Beta vulgaris root explants. Nematologica 15: 129-143. 8. Moriarty, F. 1964. The monoxenic culture of beet eelworm (Heterodera schachtii Schm.) on excised roots of sugar beet (Beta vulgaris L.). Parasitology 54: 289-293. 9. Mugnier, J. 1988. Transport of the nematicide oxamyl in roots transformed with Agrobacterium rhizogenes. Supplement to the Journal of Nematology, Annals of Applied Nematology 2:29-33.
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10. M/iller, J. 1978. Monoxenic culture of Heterodera schachtii on cruciferous plants and its use for selection of resistant host plants. Revue de N6matologie 1:47-52. 11. McDonnell, R. E., R. D. Clark, W. A. Smith, and M. A. Hinchee. 1987. A simplified method for the detection of neomycin phosphotransferase II activity in transformed plant tissues. Plant Molecular Biology Reporter 5:380-386. 12. Murashige, T., and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15:473-487.
13. Paul, H., C. Zijlstra, J. E. Leeuwangh, F. A. Krens, and H.J. Huizing. 1987. Reproduction of the beet cyst nematode Heterodera schachtii Schm. on transformed root cultures of Beta vulgaris L. Plant Cell Reports 6:379-381. 14. Verdejo, S., B. A. Jaffee, and R. Mankau. 1988. Reproduction of Meloidogynejavanica on plant roots genetically transformed by Agrobacterium rhizogenes. Journal of Nematology 20:599-604.
